Using Two Retrotransposon Based Marker Systems ( IRAP and REMAP ) for Molecular Characterization of Olive ( Olea europaea L . ) Cultivars

Olive (Olea europaea L.) is one of the most characteristic agricultural trees of the Mediterranean region and has a large number of cultivar diversity. Olive cultivar characterization is very important especially for the fruit productivity and olive oil quality. In the present study, 46 clones belonging to Turkey (eight cultivars, each having five clones) and Italy (two cultivars, each having three clones) were assessed for cultivar characterization via inter-retrotransposon amplified polymorphism (IRAP) and retrotransposon-microsatellite amplified polymorphism (REMAP) marker systems using 10 LTR and 10 ISSR primers. In total, 368 band profiles were obtained, 358 of which are polymorphic (97.28% polymorphism). The cultivars were segregated into three main groups, each group having several branches, where all the clones of each cultivar were belonging to the same main group. The only exception to that was the distribution of the clones of cultivar Yaglik, ‘Yaglik 4’ and ‘Yaglik 5’, into different main groups. IRAP and REMAP analysis showed a high level of genetic variability among the olive cultivars in this study and this marker systems would be useful tool for clonal selection programs.


Introduction
Olive (Olea europea L.) has more than 2600 cultivars, and has been cultivated since the ancient times in the Mediterranean area, where it is still the most significant oil-producing crop, the region accounts for not less than 97% of the world production and 91% of world consumption of olive oil (Luchetti, 1993;Rugini and Lavee, 1992;Zohary and Hopf, 1994).The cultivated form of olive (O.europaea L. var.europaea) is produced from the seedlings of wild form of olive (O.europaea L. var.sylvestris) by cutting or grafting (Green, 2002), where these two interfertile olive forms produce a large number of varieties with high levels of heterozygosity and genetic diversity among predominantly allogamus cultivars (Angiolillo et al., 1999;Diaz et al., 2006).This variability in olive cultivars makes the cultivar identification extremely difficult, which is actually crucial for the determination of olive productivity and oil quality, i.e., properties inherited to a variety (Fiorino and Rallo, 1999).In this respect, molecular markers are very useful for characterization of olive varieties and detection of synonymous and homonymous.Indeed, wide range of DNA molecular marker types have been used for genetic variability and cultivar identification of olive during the last ten years such as RAPDs (Hess et al., 2000;  the plant kingdom.Non-LTR retrotransposons defect terminal repeats and encode proteins with significantly less similarity to those of the retroviruses (Agarwal et al., 2008).
IRAP and REMAP marker systems, in contrast to other techniques, characterize large genetic dissimilaries in the cultivars.Integration of retrotransposon creates new links between genomic DNA and their conserved ends, for this reason, they can be used as useful molecular markers.Retrotransposon-based marker systems are an important source of plant genetic diversity and this system mostly use PCR to reproduce a segment of genomic DNA at this link (Kalendar and Schulman, 2006).Therefore, genetic differentiation perseveres through the old copies, but insertion of new copies arises.The ancestral and reproduced typical locus differentiations become potential as the lack of the introduced sequence can be, with high reliance, conceived ancestral.Basicly, the presence of a fixed retrotransposon in relevant taxa recommends their orthologues integration while the lack of specific elements shows the plesiomorphic condition prior to integration in more different taxa.A phylogenetic tree of species based on the presence of retrotransposons dispersion and its irreversible facts during evolution can build this presence/absence analyses.This is why retrotransposons are accepted to show strong synapomorphies (Shedlock and Okada, 2000).
There are only few reports available on application of retrotransposon-based marker systems for molecular identification in olive.The first study, reported by Hernandez et al. (2001), presents the first evidence of a retrotransposon-like element in olive using SCAR-markers.Giordani et al. (2004) and Koksal et al. (2014) reported genetic diversity in olive cultivars using retrotransposon-based marker system, as well.They used the IRAP (Koksal et al, 2014) and REMAP (Giordini et al., 2004) marker systems for molecular characterisation of olive cultivars.These reports can be considered as the first comprehensive research, where retrotransposon-based marker technique is used on olive genome.However, these short presentations have not generated yet a published research article.

DNA Extraction
The total genomic DNA was extracted by using CTAB method (Doyle and Doyle, 1987) after grinding the young leaf tissue to a fine powder.DNA sample concentration was determined using a nanodrop spectrophotometer (BioSpec-nano; Shimadzu-Biotech).DNA samples were diluted to 50 ng/µl prior to IRAP and REMAP PCR amplifications.

REMAP (Retrotransposon-Microsatellite Amplified Polymorphism) PCR
REMAP-PCR DNA amplification was performed using a combination of 10 LTR primers (0.2 mM for each reaction) and 10 ISSR primers, each primer at the concentration of 0.2 mM for each reaction (Martins-Lopes et al., 2009;Smykal et al., 2011; Table 2).Amplification conditions and separating were the same as for IRAP PCRs (see above).DNA fragments of IRAP and REMAP PCRs were scored by their presence (1) or absence (0), and the ones at low intensities were scored only if they were reproducible in both the PCR runs.Cluster analysis was performed to construct dendrograms, with the unweighted pair-group method by arithmetic averages (UPGMA) from the similarity data matrices using Jaccard's coefficient (D-UPGMA, 2002).

Results and Discussion
Molecular fingerprinting of forty six clones belonging to ten cultivars was carried out using IRAP and REMAP analysis, and very high polymorphism (97.28%, in average) was detected by both the methods.The total of 368 reproducible bands, ranging from 125 to 3600 bp, were scored.126 bands were obtained by IRAP and 242 were by REMAP techniques, with a similar polymorphism rates of 96.82% (122 polymorphic bands) and 97.52% (236 polymorphic bands), respectively.The highest polymorphism rate was obtained by REMAP PCR 2 amplification, and produced 23 polymorphic bands (Fig. 1).
Although the two marker systems produced different cluster numbers in all cultivars according to the dendrogram analyses, high compatibility was obtained from both and their polymorphism rate was very similar (96.82% for IRAP and 97.52% for REMAP).The high level of polymorphism was detected with B1-5, Balıkesir cv.'Edincik' by both the two marker systems; indeed this cultivar was very distant from the others and was grouped into different cluster (it cultivars 'Canino' and 'Frantoio' (similarity ranges from 0.245 to 0.379).On the contrary, B1-5, Balıkesir cv.'Edincik' (Cluster I) indicated independent branches from the other cultivars (Fig. 1) and this cultivar had many polymorphic bands in the most of PCR gel analysis (Fig. 5A).On the other hand, the combined dendrograms indicated that clones Y1, 2 and 3 (Cluster III) and Y4 and 5 (Cluster IV) of cv.'Yaglik' were in different groups.This was not surprising as there were many polimorfic bands in PCR gel analysis (Fig. 5B).was seen in "Cluster I" for three dendrograms).This cultivar is very different from the others also for the morphological characteristics; it has relatively bigger fruits, low oil and high water content (Isik et al., 2011).
The dendrogram analyses almost fully matched with same clones, however there was some evidence for clustering of clones derived from different branches.Clone (cv.'Yaglik') Y1-3 and Y4-5 were in different groups and their similarity ranges were between 0.419 and 0.480.These differences could be the result of crosspollination with local populations (Contento et al., 2002), somatic mutations (Belaj et al., 2004), and sometimes could also be due to the presence of a high level of homonymy in the collection.This is a significant problem and is a great risk for olive producers, as the renewal of certified orchards should be based on certified plants (Gemas et al., 2004;Martins-Lopes et al., 2007;Hannachi et al., 2008).However, classical olive certification system is based on morphological and agronomic procedures, which are affected by the environmental conditions, and mislabeling accessions can negatively affect certification of olive products (Hannachi et al., 2008).Molecular marker systems are of great importance to overcome such problem, and is necessary to determine the polymorphism level of olive cultivars and homonymy and synonym problems in olive germplasm.High values of observed heterozygosity were recorded for all the IRAP and REMAP markers investigated.
Determination of genetic relationships among cultivars eases efficient sampling, operating and using of germplasm resources.In the present study, IRAP and REMAP analysis displayed a high level of genetic variability among olive cultivars, indicating a potential resource for the use of this germplasm in clonal selection programs.